Intraclonal Diversity in Follicular Lymphoma Analyzed by Quantitative Ultradeep Sequencing of Noncoding Regions

Abstract

Cancers are characterized by genomic instability and the resulting intra-clonal diversity is a prerequisite for tumor evolution. Therefore, metrics of tumor heterogeneity may prove to be clinically meaningful. Intra-clonal heterogeneity in follicular lymphoma (FL) is apparent from studies of somatic hypermutation (SHM) caused by Activation Induced Deaminase (AID) in IGH. Aberrant SHM (aSHM), defined as AID activity outside of the IG loci, predominantly targets non-coding regions causing numerous “passenger” mutations but has the potential to generate rare significant “driver” mutations. The quantitative relationship between SHM and aSHM has not been defined. To measure SHM and aSHM, ultradeep sequencing (>20,000 fold coverage) was performed on IGH (∼1650nt) and 9 other non-coding regions potentially targeted by AID (combined 9411nt), including the 5′UTR of BCL2. Single nucleotide variants (SNV) were found in 12/12 FL specimens (median 136 SHM and 53 aSHM). The aSHM SNVs were associated with AID-motifs (p<0.0001). The number of SNVs at BCL2 varied widely among specimens and correlated with the number of SNVs at 8 other potential aSHM sites. In contrast SHM at IGH was not predictive of aSHM. Tumor heterogeneity is apparent from SNVs at low variant allele frequencies (VAF); the relative number of SNVs with VAF<5% varied with clinical grade indicating that tumor heterogeneity based on aSHM reflects a clinically meaningful parameter. These data suggest that genome-wide aSHM may be estimated from aSHM of BCL2 but not SHM of IGH. The results demonstrate a practical approach to the quantification of intra-tumoral genetic heterogeneity for clinical specimens.

Ultradeep analysis of tumor heterogeneity in regions of somatic hypermutation

“…We found that BCL2 , followed by BCL6 , showed the most consistent and differential aSHM signal in FL specimens, with SNVs in 12/12 FL cases and 0/3 in the reactive LN controls. The range of SNVs in BCL2 varied widely, from 2 to 101 per patient; raising the possibility that overall BCL2 aSHM rate might be clinically informative through risk stratification [ 54 ]. BCL6 shows a similar pattern, but with a much lower overall aSHM rate, in the range of 2 to 39 SNV per patient.…”

“…The frequencies associated with MFC varied from 10% to 45% (median of 20%) and in all but one case (FL-134) the majority of SNVs in any given tumor had frequencies consistent with their current MFC. Several FL specimens (5/12) had no SNVs outside the MFC; three with a low overall mutation rate (<0.7%) and two with a different type of FL (Grade 3 vs. Grades 1 and 2), suggestive of a possible biological aspect to this finding [ 54 ]. Of the remaining FL specimens, the vast majority (over 80%) of the SNVs outside the MFC were found at ≤3%, while over half were ≤1% (see Additional file 4 : Table AF1).…”

Immunoglobulin rearrangement analysis from multiple lesions in the same patient using next‐generation sequencing

“…Next‐generation sequencing (NGS) of immunoglobulin heavy locus (IGH) gene rearrangements for the monitoring of minimal residual disease in patients with leukaemia or myeloma has already been proved useful . In molecular pathology, where formalin‐fixed paraffin‐embedded (FFPE) tissues are frequently used, resulting in suboptimal DNA quality, IG‐rearrangement assessment has different requirements and has only just begun . In the current study, we have investigated semi‐conductor sequencing using the Ion Torrent Personal Genome Machine (Ion PGM) for IG rearrangement assessment in pathology.…”

“…[6][7][8][9][10][11][12][13] In molecular pathology, where formalinfixed paraffin-embedded (FFPE) tissues are frequently used, resulting in suboptimal DNA quality, IG-rearrangement assessment has different requirements and has only just begun. 14,15 In the current study, we have investigated semi-conductor sequencing using the Ion Torrent Personal Genome Machine (Ion PGM) for IG rearrangement assessment in pathology. Because of its short running time, 16,17 the Ion PGM is often used in molecular pathology laboratories for mutation detection in oncogenes in the context of prediction of therapy response in which a short turnaround time is essential.…”

The Common Key to Class-Switch Recombination and Somatic Hypermutation: Discovery of AID and Its Role in Antibody Gene Diversification

“…Furthermore, AID was found to be important for the IGH/ CMYC rearrangements present in some B lineage neoplasms (murine plasmacytomas, human Burkitt lymphoma, and other lymphomas) (37). More recent studies have identified numerous genes harboring oncogenic mutations that can be traced backed to AID because of their unique mutational signatures (38–42). Overall, these findings have clarified the natural history of a substantial subset of lymphomas.…”